7,12-Dimethylbenz[a]anthracene-induced mutation in the Tk gene of Tk(+/-) mice: Automated scoring of lymphocyte clones using a fluorescent viability indicator

7,12-Dimethylbenz[a]anrhracene (DMBA) is a rodent carcinogen and a potent i n vivo mutagen for the X-linked hypoxanthine guanine phosphoribosyl transfe rase (hprt) gene of rats and for the loci transgene of Big Blue mice and ra ts. Although DMBA is also a powerful clastogen, molecular analysis of these DMBA-induced hprt and loci mutations indicates that most are single base-p air (bp) substitutions and 1- to 3-bp frameshifts. In the present study, we evaluated the types of mutations induced by DMBA in the autosomal thymidin e kinase (Tk) gene of Tk(+/-) mice. Male and female 5- to 6-week-old animal s were injected i.p. with DMBA at a dose of 30 mg/kg. Five weeks after the treatment, hprt and Tk mutant frequencies were determined using a limiting dilution clonal assay in 96-well plates. We established conditions For the automated identification of wells containing expanded lymphocyte clones usi ng the fluorescent indicator alamarBlue. This procedure allowed the unbiase d identification of viable clones and calculation of mutant frequencies. In male mice, DMBA treatment increased the frequency of hprt mutants from 1.8 +/- 1.1 to 34 +/- 9 x 10(-6), and Tk mutants from 33 +/- 12 to 78 +/- 26 x 10(-6); treated female mice had a significant but lower increase in hprt m utant frequency than did males. Molecular analysis of DMBA-induced Tk mutan ts revealed that at least 75% had the entire wild-type Tk allele missing. T he results indicate that the predominant types of DMBA-induced mutation det ected by the autosomal Tk gene are different From those detected by the X-l inked hprt gene. The Tk gene mainly detects loss of heterozygosity mutation , whereas the majority of mutations previ ovsly found in the hprt gene were point mutations. Environ. Mel. Mutagen. 36.283-291, 2000. Published 2000 W iley-Liss, Inc.dagger